A glucocorticoid-receptor inactivating enzyme has been identified in and partially purified from the high-speed particulate fraction of rat liver and thymocytes. The thymocyte enzyme is inhibited by glucose-1-phosphate and the hepatic enzyme by fluoride, suggesting that the inactivation results from a dephosphorylating activity. The glucocorticoid receptor of mouse fibroblasts can be completely inactivated by incubation with highly purified alkaline phosphatase. The ability of the enzyme preparation to inactivate the specific glucocorticoid binding capacity of L cell cytosol, like its ability to dephosphorylate p-nitrophenyl phosphate, is both zinc dependent and inhibited by arsenate. Both activities co-elute on DEAE cellulose purification of the enzyme. As with the endogenous thymocyte and hepatic enzymes, it is only the unbound receptor that is inactivated. In a mixed system containing both glucocorticoid and estrogen receptors the glucocorticoid receptor is selectively inactivated by both purified alkaline phosphatase and the particulate-bound endogenous thymocyte receptor-inactivating enzyme. A portion of this work is in press in P.N.A.S.